As the numbers of patients continue to rise after high-dose chemotherapy and/ or irradiation for cancer and other diseases, more attention is focused on improving their quality-of-life, with fertility one of the top priorities for young people. Women have had fewer options for avoiding iatrogenic infertility and menopause compared with men, and prepubertal girls have virtually none. Cryopreservation or vitrification of embryos is a standard of care, and oocyte banking is now emerging for single women, but neither of these assisted reproductive technologies is universally suitable or acceptable, nor can either help children. Low temperature banking of ovarian tissue slices, and perhaps whole ovaries one day, now offers an option for these people and, so far, six full-term pregnancies have been established after transplanting thawed tissue, and several others after fresh tissue, but no grafts were still functioning after three years. This strategy for preserving fertility is still considered 'experimental', though proof of principle was established many years ago in animal models. Those studies revealed a large and variable fraction of follicles is lost by ischemia during the first 2-5 days until the grafts are revascularized, whereas the impact of cryoinjury was comparatively minor. According to our hypothesis, a reduction in ischemia time will be beneficial for extending graft function and potentiating fertility by reducing the amount of necrotic tissue and the fraction of wasted follicles. In Aim 1, we will characterize the cellular and molecular determinants of angiogenesis in ovarian grafts and their temporal course in relation to follicular survival. This aim will fill a knowledge gap by investigating the role of candidate factors, notably VEGF and PDGF, involved in revascularizing mouse ovaries grafted into skeletal muscle and human ovarian xenografts in NOD-scid mice as a translational model. The studies will include novel methods to monitor reperfusion non-invasively by microultrasound during the first two weeks post-grafting, and molecular and cellular quantification of vascular growth, angiogenic factors, follicular survival and factors in the extracellular matrix. In Aim 2, we will test two experimental strategies for improving vascular perfusion and follicle survival by tissue engineering either at the future graft site or the graft itself. In the first, we will implant alginate hydrogels into muscle for local, phased release of angiogenic factors to stimulate endothelial cells and pericytes with the goal of accelerating the formation of a stable microcirculation in murine grafts. In the second, we will reduce collagen bulk in human ovarian tissue to test whether the higher fiber density is a barrier to vascular invasion and potentially responsible for the higher fraction of follicles wasted. A successful outcome of either of these experiments could lead to clinical implementation for patients who plan to bank their ovarian tissue to preserve fertility. PUBLIC HEALTH RELEVANCE: There is an urgent public health need for effective and acceptable fertility preservation technologies to serve rising numbers of patients surviving cancer treatment after drugs and irradiation that potentially cause infertility and premature ovarian failure. Ovarian tissue banking has been adopted by several thousand patients worldwide because it offers some advantages over embryo/ oocyte banking, especially for children, but tissue grafted back has only generated a few viable pregnancies so far and only functions for a few years. This research project will fill the knowledge gap about the biology of revascularization of ovarian grafts, which limits the fraction of surviving follicles, and will test two strategies aimed at enhancing angiogenesis.